I am trying to sub-clone my insert from pcdna 3 into PBSK vector. I have cut both with Xho1 & EcoR1. The fallout from pcdna3 (1.2Kbp) & pbsk vector backbone extracted from gel separately. Subsequent ligation reaction when I transformed into DH5alpha competent cell with amp+ media . I got no colonies. I have repeated the experiment but still the same result please suggest..
Did you check whether EcoRI and XhoI cuts somewhere else in your PBSK vector backbone?
If there is no restriction site for either one of these enzymes. The most likely reason might be your ligation didn't work. I usually take 100ng of the gel excised enzyme digested vector and calculate the amount of insert need using the https://nebiocalculator.neb.com/#!/ligation neb calculator. Leave the ligation for either 2 hours at room temperature or 16 degree celsius for o/n (Much better than room temperature). Use 3-5 microliter of ligation reaction for my transformation. After the heat shock incubate the bacteria at 37 degree celsius with shaking in the LB/SOC media with out any antibiotics for 60-80 min. Then Plate them on the selection plate. Please check in your case whether PBSK vector has ampicillin marker in it.
Thank You Chandhuru Jagadeesan. After double digestion of PBSK vector with Xho1 & EcoR1 I got 2 bands one of 3kb size and the other was its previously cloned insert of 2.5kb size which was flanked by xho1 and ecoR1..same with the pcdna3 my GOI which i have to clone is flanked by Xho1 and EcoR1 ..
Seems like the restriction is done correctly. I agree with Chandhuru that ligation might be your problem. His protocol sounds good. I normally add 10 µL ligation reaction to 100 µL cells.
Alternatively you competent cells might be the source of trouble. Did you check their transformation efficiency?
Thank You Christiane . I repeated my transformation again but this time i took different competent cells not the same i have been using and guess what ? VOILA!! IT WORKED . I got colonies . Thank you christiane & Chandhuru for helping me out.
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